Werner syndrome (WS) is an "adult-onset" genetic disorder characterized by multiple features consistent with accelerated aging (i.e., a "segmental progeroid syndrome"). An aged appearance and common age-related disorders such as type 2 diabetes mellitus often emerge during the early 20s. Bilateral ocular cataracts are typically extracted during the early 30s, and atherosclerosis and cancers occur by the fifth decade. "Classical" WS is caused by mutations at the WRN gene, which encodes a DNA helicase/exonuclease. Clinically diagnosed or suspected WS cases are referred to our International Registry of Werner Syndrome (University of Washington, Seattle WA) from all over the world for molecular diagnosis. Cases without WRN mutations are operationally categorized as "atypical WS" (AWS). In 2003, we identified LMNA mutations among a small subset of atypical WS cases by a candidate gene approach. As of January 2008, the Werner registry has 107 WS pedigrees with WRN mutations, 6 AWS patients with LMNA mutations, and 41 AWS pedigrees in which we did not find WRN or LMNA mutations. Primary fibroblasts, lymphoblastoid cell lines or both are available from 29 of the 41 atypical cases. The purpose of this study is to identify additional genes responsible for AWS, perhaps revealing a single heretofore unsuspected major pathogenic locus. Our approach is motivated by the knowledge that ~one-third of disease mutations are thought to result in premature termination codons that lead to the rapid degradation of mRNA via the nonsense-mediated mRNA decay (NMD) pathway. A strategy using inhibition of NMD combined with array analysis has been developed to identify disease genes. In this approach, mutated mRNAs that would have undergone NMD are stabilized and their mRNAs detected by the array analysis. Our screens will utilize three different NMD inhibitors, emetine (inhibits initial scanning of truncated proteins), caffeine (inhibits SMG1 kinase in the NMD complex combined with actinomycin D), and an RNAi against UFP1 (a component of the NMD complex) (Aim 1). Stabilization of mutant mRNAs will be confirmed by quantitative PCR (Aim 2), and candidate genes will be sequenced using DNA from the index cases and other AWS cases (Aim 3). Once the genetic evidence is established, further investigations of pathogenesis of will be proposed via separate funding. PUBLIC HEALTH RELEVANCE: Atypical Werner syndrome patients present accelerated aging phenotypes similar to Werner syndrome but do not carry WRN or LMNA mutations. We proposed to identify the genes responsible for atypical WS using the strategy of non-sense mediated mRNA decay inhibition and array analysis.